This invention generally relates to the purification and characterization of heparinase I, II, and III from Flavobacterium heparinum and antibodies thereto.
Heparin and heparan sulfate represent a class of glycosaminoglycans characterized by a linear polysaccharide of D-glucosamine (1.fwdarw.4) linked to hexuronic acid (Linhardt, R. J. (1991) Chem. Ind. 2, 45-50; Casu, B. (1985) Adv. Carbohydr. Chem. Biochem. 43, 51-134). Heparin and heparan sulfate are complex carbohydrates that play an important functional role in the extracellular matrix of mammals. These polysaccharides modulate and regulate tissue level events that take place either during development under normal situations or wound healing and tumor metastasis under pathological conditions.
Much of the current understanding of heparin and heparan sulfate sequence has relied on studies of their biosynthesis (Linhardt, R. J., Wang, H. M., Loganathan, D., and Bae, J. H. (1992) Biol. Chem. 267, 2380-2387; Lindahl, U., Feingold, D., and Roden, L. (1986) Trends Biochem. Sci. 11, 221-225; Jacobson, I., and Lindahl U. (1980) J. Biol. Chem. 255, 5094-5100; Lindahl, U., and Kjellen, L. (1987) in The Biology of Extracellular Matrix Proteoglycans (Wight, T. N., and Mecham R., eds) pp. 59-104, Academic Press, New York). Recent efforts (Linhardt, R. J., Rice, K. G., Kim, Y. S., Lohse, D. L., Wang, H. M., and Loganathan, D. (1988) Biochem. J. 254, 781-787; Linhardt, R. J., Turnbull, J. E., Wang, H. M., Loganathan, D., and Gallagher, J. T. (1990) Biochemistry 29, 2611-2617) have focused on the application of enzymatic methods to depolymerize these complex polysaccharides into oligosaccharides that could then be structurally characterized (Linhardt, et al. (1992) Biol. Chem. 267, 2380-2387; Linhardt, et al., (1988) Biochem. J. 254, 781-787; Loganathan, D., Wang, H. M., Mallis, L. M., and Linhardt, R. J. (1990) Biochemistry 29, 4362-4368).
Enzymatic methods for heparin and heparan sulfate depolymerization are very specific and require mild conditions giving oligosaccharide products that closely resemble the glycosaminoglycans from which they were derived. Two types of enzymes that degrade heparin and heparan sulfate glycosaminoglycans are the polysaccharide lyases from prokaryotic sources that act through an eliminative mechanism (Linhardt, R. J., Galliher, P. M., and Cooney, C. L. (1986) Appl. Biochem Biotech. 12, 135-176), and the glucuronidases (hydrolases) from eukaryotic sources that act through a hydrolytic mechanism.
Prokaryote degradation of heparin and heparan sulfate has primarily been studied using enzymes derived from Flavobacterium heparinum (Linker, A., and Hovingh, P. (1965) J. Biol. Chem. 240, 3724-3728; Linker, A., and Hovingh, P. (1970) J. Biol Chem. 245, 6170-6175); Dietrich, C. P., Silva, M. E., and Michelacci, Y. M. (1973) J. Biol. Chem. 248, 6408-6415; Silva, M. E., Dietrich, C. P., and Nader, H. B. (1976) Biochem. Biophys. Acta 437, 129-141). This bacterial degradation begins with the action of three (or possibly more) eliminases. These heparin lyases produce oligosaccharides with .DELTA..sub.4,5 -unsaturated uronic acid residues a their non-reducing termini. These eliminases probably act in concert to convert heparin and heparan sulfate to disaccharides.
Heparin lyases are a general class of enzymes that are capable of specifically cleaving the major glycosidic linkages in heparin and heparan sulfate. Three heparin lyases have been identified in Flavobacterium heparinum, a heparin-utilizing organism that also produces exoglycuronidases, sulfoesterases, and sulfamidases that further act on the lyase-generated oligosaccharide products (Yang, V. C., Linhardt, R. J., Berstein, H., Cooney, C. L., and Langer, R. (1985) J. Biol. Chem. 260, 1849-1857; Galliher, P. M., Linhardt, R. J., Conway, L. J., Langer, R., and Cooney, C. L. (1982) Eur. J. Appl. Microbiol. Biotechnol. 15, 252-257). These lyases are designated as heparin lyase I (heparinase, EC 4.2.2.7), heparin lyase II (heparinase II, no EC number) and heparin lyase III (heparitinase EC 4.2.2.8). Although the specificities of these enzymes are not completely known, studies using partially purified enzymes with heparin, heparan sulfate, and structurally characterized heparin oligosaccharides have led to an understanding of the linkages susceptible to enzymatic cleavage (Lindhardt, et al., (1990), Lohse (1992), Rice, K. G., and Linhardt, R. J. (1989) Carbohydr. Res. 190, 219-233). The three purified heparin lyases differ in their capacity to cleave heparin and heparan sulfate: Heparin lyase I primarily cleaves heparin, heparin lyase III specifically cleaves heparan sulfate and heparin lyase II acts equally on both heparin and heparan sulfate (Linhardt, et al., 1986; Linhardt, et al., 1990).
Several Bacteroides sp. (Saylers, A. A., Vercellotti, J. R., West, S.E.H., and Wilkins, T. D. (1977) Appl. Environ. Microbiol. 33, 319-322; Nakamura, T., Shibata, Y., and Fujimura, S. (1988) J. Clin. Microbiol. 26, 1070-1071) also produce heparinases, however, these enzymes are not well characterized. A heparinase has also been purified to apparent homogeneity from an unidentified soil bacterium (Bohmer, L. H., Pitout, M. J., Steyn, P. L., and Visser, L. (1990) J. Biol. Chem. 265, 13609-13617). This enzyme differs from those isolated from Flavobacterium heparinum in its molecular weight (94,000), pI (9.2), amino acid composition and kinetic properties (K.sub.m of 3.4 .mu.M and V.sub.max of 36.8 .mu.mol/min, pH optimum of 7.6).
Three other heparin lyases, partially purified from Flavobacterium sp. Hp206, have molecular weights of 64,000, 100,000, and 72,000, as reported by Yoshida, K., Miyazono, H., Tawada, A., Kikuchi, H., Morikawa, K., and Tokuyasu, K. (1989) 10th Annual Symposium of Glycoconjugates, Jerusalem, different from heparin lyases I-III.
The heparin lyases of F. heparinum are the most widely used and best studied (Lindhardt, (1986)). Linker and Hovingh (1970) first separated these lyase activities, fractionating a crude lyase fraction into a heparinase (heparin lyase I) and a heparitinase (heparin lyase III). Both activities were purified by 50-100-fold, but no physical characterization of these enzymes was performed.
Dietrich and co-workers (Dietrich, et al., 1973); Silva, et al., (1976); Silva, M. E., and Dietrich, C. P. (1974) Biochem. Biophys. Res. Commun. 56, 965-972; Michelacci, Y. M., and Dietrich, C. P. (1974) Biochem. Biophys. Res. Commun. 56, 973-980) and Ototani and Yosizawa (Ototani, N., and Yosizawa, Z. (1978) J. Biochem. (tokyo) 84, 1005-1008; Ototani, N., and Yosizawa, Z. (1979) Carbohydr. Res. 70, 295-306; Ototani, N., Kikiuchi, M., and Yosizawa, Z. (1981) Carbohydr. Res. 88, 291-303; Ototani, N., and Yosizawa, Z. (1981) Proceedings of the 6th International Symposium on Glycoconjugates, pp. 411-412, September 20-25, Tokyo, Japan Scientific Press, Tokyo) isolated three lyases, a heparinase (heparin lyase I) and two heparitinases, from F. heparinum. The heparinase acted on heparin to produce mainly trisulfated disaccharides (Dietrich, C. P., and Nader, H. B. (1974) Biochem. Biophys. Acta 343, 34-44; Dietrich, C. P., Nader, H. B., Britto, L. R., and Silva, M. E. (1971) Biochem. Biophys. Acta 237, 430-441); Nader, H. B., Porcionatto, M. A., Tersariol, I.L.S., Pinhal, M. S., Oliveira, F. W., Moraes, C. T., and Dietrich, C. P. (1990) J. Biol. Chem. 265,16807-16813) purified two heparitinases (called heparitinase I and II, possibly corresponding to heparin lyases II and III, although no physical properties of these enzymes were presented) and characterized their substrate specificity toward heparin and heparan sulfate. Heparitinase I degraded both N-acetylated and N-sulfated heparan sulfate while heparitinase II degraded primarily N-sulfated heparan sulfate.
McLean and Co-workers described the specificity of a partially purified heparinase II (Moffat, C. F., McLean, M. W., Long, W. F., and Williamson, F. B. (1991) Eur. J. Biochem. 197, 449-459; McLean, M. W., Long, W. F., and Williamson, F. B. (1985) in Proceedings of the 8th International Symposium on Glycoconjugates, pp. 73-74, September, Houston, Paeger Publishers, New York; McLean, M. W., Bruce, J. S., Long, W. F., and Williamson, F. B. (1954) Eur. J. Biochem. 145, 607-615). Although no evidence of homogeneity or any physical properties for heparinase II were presented, the broad specificity on various polymeric substrates (Moffat, et al., (1991)) identifies the enzyme as heparin lyase II (Lindhardt, et al., (1990); McLean, et al., (1985).
Linhardt et al. (1984) Appl. Biochem. Biotech. 9, 41-55) reported the purification of heparinase (heparin lyase I) to a single band on SDS-PAGE. Affinity purification of heparin lyase I on heparin-Sepharose failed, apparently due to degradation of the column matrix. Sufficient quantities of pure heparin lyase I for detailed characterization studies and amino acid analysis were first prepared by Yang et al. (1985). Heparin lyase I was used to prepare polyclonal antibodies in rabbits for affinity purification of heparin lyase I, but excessively harsh conditions required to elute the enzyme resulted in substantial loss of activity (Lindhardt, (1985)). Yang, V. C., Bernstein, H., Cooney, C. L., and Langer, R. (1987) Appl. Biochem. Biotech. 35-50)) also described a method to prepare heparin lyase I.
Seikagaku Co. has recently orally reported the molecular weights of their commercial enzymes corresponding to heparin lyase I-III to be 43,000, 84,000, and 70,000, respectively (Yoshida, K. (1991) International Symposium on Heparin and Related Polysaccharides, September 1-6, Uppsala, Sweden). These reports are in close agreement to the molecular weights described herein, but no details of their purification or characterization methods have been published.
Heparin lyases have been used to establish the presence of heparin in mixtures of proteoglycans (Kanwar, Y. S., and Farguhar, M. G. (1979) Presence of heparan sulfate in the glomerular basement membrane. Proc. Natl. Acad. Sci., USA 76, 1303-1307), to depolymerize heparin and heparan sulfate to characterize the structure of the resulting oligosaccharides (Linhardt, R. J., Loganathan, D. A1-Hakim, A., Wang, H.-M., Walenga, J. M., Hoppensteadt, D., and Fareed, J. (1990) Oligosaccharide mapping of low molecular weight heparins: structure and activity differences. J. Med. Chem. 33, 1639-1645; Linhardt, R. J., Rice, K. G., Kim, Y. S., Lohse, D. L., Wang, H. M., and Loganathan, D. (1988). Mapping and quantification of the major oligosaccharide components of heparin. Biochem. J. 254, 781-787; Merchant, Z. M., Kim, Y. S., Rice, K. G., and Linhardt, R. J. (1985). Structure of heparin-derived tetrasaccharides. Biochem. J. 229, 369-377; Turnbull, J. E., and Gallagher, J. T. (1988) Oligosaccharide mapping of heparan sulphate by polyacrylamide-gradient-gel electrophoresis and electrotransfer to nylon membrane. Biochem J. 251, 597-608), to produce low molecular weight heparin preparations with anticoagulant and complement inhibitory activities (Linhardt, R. J., Grant, A., Cooney, C. L., and Langer, R. (1982) Differential anticoagulant activity of heparin fragments prepared using microbial heparinase. J. Biol. Chem. 257, 7310-7313; Linhardt, R. J., and Loganathan, D. (1990a). Heparin, heparinoids and heparin oligosaccharides: structure and biological activity. In C. G. Gebelein (Ed.), Biomimetic Polymers (pp. 135-173). New York: Plenum Press; Sharath, M. D., Merchant, Z. M., Kim, Y. S., Rice, K. G., Linhardt, R. J., and Weiler, J. M. (1985) Small heparin fragments regulate the amplification pathway of complement. Immunopharmacology 9, 73-80) and to remove heparin from the circulation (Langer, et al., 1982). Heparin depolymerising enzymes are excellent tools to understand the role of heparin-like molecules in the extracellular matrix or to be used in different tissue microenvironments to modulate and alter the extracellular matrix in a highly specific manner. However, studies utilizing heparin lyases are hampered by difficulties in purifying the enzymes from Flavobacterium heparinum, especially with regard to separation of the three enzymes from each other (Linhardt, et al., 1985). Specifically, the capacity of heparin lyase II to cleave both heparin and heparan sulfate makes it difficult to distinguish from heparin lyase I which cleaves heparin and heparin lyase III which cleaves heparan sulfate.
Although all three of these heparin/heparan sulfate lyases are widely used, with the exception of heparin lyase I, there is no information on the purity or physical and kinetic characteristics of heparinase II and heparinase III. The absence of pure heparin lyases, resulting in ambiguities with respect to substrate specificity. This is due to contamination of other lyases in the preparation, and a lack of understanding of the optimal catalytic conditions and substrate specificity has stood in the way of the use of these enzymes as reagents for the specific depolymerization of heparin and heparan sulfate into oligosaccharides for structure and activity studies, and for use in clinical studies.
It is therefore an object of the present invention to provide a method for purification and characterization of heparinase I, heparinase II, and heparinase III.
It is a further object of the present invention to provide purified and characterized heparinase I, heparinase II, and heparinase III.
It is a still further object of the present invention to provide the conditions for optimal use and peptide map of the purified heparinase II and heparinase III.
It is another object of the present invention to provide the amino acid compositions of the three heparinases.
It is another object of the present invention to provide antibodies for heparinase I, II, and III which can be used in the purification and characterization of heparinases.